Supplementary Material for: Mesenchymal Stromal Cells from Dental Tissues Demonstrate Neuronal Potential Pre- and Post-Induction

Introduction: Mesenchymal stromal cells (MSCs) play a crucial role in tissue repair and exhibit anti-inflammatory properties, making them promising for regenerative medicine. Dental tissue-derived MSCs, such as stromal cells from human exfoliated deciduous teeth (SHEDs) and dental pulp stromal cells (DPSCs), express neural markers and hold the potential for treating neurodegenerative diseases. Nonetheless, their relative ability to differentiate into neurons is still unclear. Given their shared embryonic origin, we hypothesised that SHEDs and DPSCs possess similar potential for neuronal differentiation along with intrinsic expression of neuronal markers. The objective of this study was to compare their differentiation abilities under standardised conditions and evaluate neuronal markers pro- and post-neuronal induction. Objective: To compare the neuronal differentiation potential of SHEDs and DPSCs. Methodology: SHEDs (n=3) and DPSCs (n=3) were collected with ethical approval, cultured, and characterised according to established MSC criteria. Clonogenicity, proliferation, senescence, and trilineage differentiation were assessed. Neuronal differentiation was induced for 21 days and evaluated using flow cytometry (SRY-Box Transcription Factor 1 (SOX1), SRY-Box Transcription Factor 2 (SOX2), Glial fibrillary acidic protein (GFAP), doublecortin, nestin, CD56, CD146), immunofluorescence for βIII-tubulin, and reverse transcription polymerase chain reaction (RT-PCR) for tubulin 3 (TUB3) and microtubule-associated protein 2 (MAP2). Results: Both SHEDs and DPSCs exhibited MSC characteristics. SHEDs showed higher clonogenicity. Early neuronal markers (e.g., SOX1, Nestin, GFAP, βIII-tubulin) were detected pre- and post-induction in both cell types without significant intergroup differences. No significant expression of TUB3 and MAP2 was observed. Conclusion: SHEDs and DPSCs show comparable neuronal marker expression profiles, suggesting similar early neuronal differentiation potential. These findings support using undifferentiated SHEDs and DPSCs in neuro-regenerative strategies, offering cost-effective and safer alternatives to pre-differentiated cells.